The objective of this proposal is the identification of immunogenic and functional domains of exotoxin A from Pseudomonas aeruginosa. To accomplish this, a library of monoclonal antibodies will be utilized as site-specific probes for the detailed analysis of this protein. We have produced a number of monoclonal reagents, some of which inhibit specific functions of the exotoxin molecule. It is our intention to apply these antibodies to the determination of: 1) the receptor-specific epitope(s) of the toxin; 2) the NAD-dependent ADP-ribosyltransferase active site of the toxin; 3) the location of the immunogenic epitopes required for the development of host immunity to toxin. The expected significance of this project: The project outlined above has very specific objectives, but the potential applications of the expected results are numerous and significant. First, a detailed knowledge of functional regions of this protein will allow an in-depth analysis of the correlation between structure and function for microbial toxins, since the complete amino acid sequence of this protein is known. Furthermore, the data obtained from this study will be used to determine the nature of the molecular lesion in a non-functional toxin produced by a mutant strain of P. aeruginosa, since the mutant and native toxins can be distinguished using mono-specific antibodies. Determination of the location and nature of the lesion in the mutant toxin (which alters the ability of the toxin to inhibit protein synthesis) will provide the basis for the production of a genetically-engineered strain designed to produce a non-functional, but immunologically cross-reactive toxin suitable for vaccine development. Additionally, knowledge of the specific immunogenic epitopes of the toxin will provide the basis for the development of synthetic peptides corresponding to regions known to elicit the formation of anti-toxin in vivo. As such, the system described (a lethal toxin well-characterized with respect to susceptible cell lines and animal model systems) will provide the means to effectively test the plausibility of the use of synthetic peptides in vaccine development.